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• W2000788954 abstract "Calreticulin is an endoplasmic reticulum Ca2+-storage protein, which influences gene expression and cell adhesion. In this study, we show that calreticulin induces fibronectin gene expression and matrix deposition, leading to differences in cell spreading and focal adhesion formation in cells differentially expressing calreticulin. We further show that these effects of calreticulin occur via a c-Src-regulated pathway and that c-Src activity is inversely related to calreticulin abundance. Since c-Src is an important regulator of focal contact turnover, we investigated the effect of c-Src inhibition on cells differentially expressing calreticulin. Inhibition of c-Src rescued the poorly adhesive phenotype of the calreticulin-underexpressing cells in that they became well spread, commenced formation of numerous focal contacts, and deposited a rich fibronectin matrix. Importantly, we show that c-Src activity is dependent on releasable Ca2+ from the endoplasmic reticulum, thus implicating Ca2+-sensitive pathways that are affected by calreticulin in cell-substratum adhesion. We propose that calreticulin affects fibronectin synthesis and matrix assembly via the regulation of fibronectin gene expression. In parallel, calcium-dependent effects of calreticulin on c-Src activity influence the formation and/or stability of focal contacts, which are instrumental in matrix assembly and remodeling. Calreticulin is an endoplasmic reticulum Ca2+-storage protein, which influences gene expression and cell adhesion. In this study, we show that calreticulin induces fibronectin gene expression and matrix deposition, leading to differences in cell spreading and focal adhesion formation in cells differentially expressing calreticulin. We further show that these effects of calreticulin occur via a c-Src-regulated pathway and that c-Src activity is inversely related to calreticulin abundance. Since c-Src is an important regulator of focal contact turnover, we investigated the effect of c-Src inhibition on cells differentially expressing calreticulin. Inhibition of c-Src rescued the poorly adhesive phenotype of the calreticulin-underexpressing cells in that they became well spread, commenced formation of numerous focal contacts, and deposited a rich fibronectin matrix. Importantly, we show that c-Src activity is dependent on releasable Ca2+ from the endoplasmic reticulum, thus implicating Ca2+-sensitive pathways that are affected by calreticulin in cell-substratum adhesion. We propose that calreticulin affects fibronectin synthesis and matrix assembly via the regulation of fibronectin gene expression. In parallel, calcium-dependent effects of calreticulin on c-Src activity influence the formation and/or stability of focal contacts, which are instrumental in matrix assembly and remodeling. Calreticulin, a Ca2+-binding protein of the endoplasmic reticulum (ER), 3The abbreviations used are: ER, endoplasmic reticulum; [Ca2+]ER, concentration of Ca2+ within the endoplasmic reticulum; [Ca2+]c, concentration of calcium in the cytosol; ECM, extracellular matrix; FAK, focal adhesion kinase; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid. has been shown to be involved in a great number of cellular processes (1.Bedard K. Szabo E. Michalak M. Opas M. Int. Rev. Cytol. 2005; 245: 91-121Crossref PubMed Scopus (126) Google Scholar). It is an important chaperone, working in conjunction with calnexin and protein disulfide isomerase; it affects intracellular Ca2+ homeostasis via its Ca2+ storage capacity and its effects on both the SERCA pumps and inositol 1,4,5-trisphosphate receptors (2.John L.M. Lechleiter J.D. Camacho P. J. Cell Biol. 1998; 142: 963-973Crossref PubMed Scopus (181) Google Scholar, 3.Camacho P. Lechleiter J.D. Cell. 1995; 82: 765-771Abstract Full Text PDF PubMed Scopus (200) Google Scholar). Calreticulin has been shown to affect cell adhesion via the induction of vinculin and N-cadherin expression and its involvement in β-catenin-associated pathways (4.Opas M. Szewczenko-Pawlikowski M. Jass G.K. Mesaeli N. Michalak M. J. Cell Biol. 1996; 135: 1913-1923Crossref PubMed Scopus (109) Google Scholar, 5.Fadel M.P. Dziak E. Lo C.M. Ferrier J. Mesaeli N. Michalak M. Opas M. J. Biol. Chem. 1999; 274: 15085-15094Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Finally, calreticulin overexpression causes a decrease in total cellular tyrosine phosphorylation levels (5.Fadel M.P. Dziak E. Lo C.M. Ferrier J. Mesaeli N. Michalak M. Opas M. J. Biol. Chem. 1999; 274: 15085-15094Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 6.Fadel M.P. Szewczenko-Pawlikowski M. Leclerc P. Dziak E. Symonds J.M. Blaschuk O. Michalak M. Opas M. J. Biol. Chem. 2001; 276: 27083-27089Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Fibronectin is a large glycoprotein which is secreted by the cell into the extracellular matrix (ECM) as a soluble dimer that, via cellular interactions, is deposited as a fibrillar meshwork that is bound to the surface of cells. Fibronectin affects the formation and the stability of cell-substratum adhesions, and conversely, cell-substratum adhesions may affect fibronectin matrix deposition (7.Katz B.Z. Zamir E. Bershadsky A. Kam Z. Yamada K.M. Geiger B. Mol. Biol. Cell. 2000; 11: 1047-1060Crossref PubMed Scopus (358) Google Scholar, 8.Geiger B. Bershadsky A. Pankov R. Yamada K.M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 793-805Crossref PubMed Scopus (1857) Google Scholar). Fibronectin assembly cannot proceed without the presence of cells (9.McDonald J.A. Annu. Rev. Cell Biol. 1988; 4: 183-207Crossref PubMed Scopus (224) Google Scholar), which implies intracellular inside-out signaling pathways that are crucial for fibronectin fibrillogenesis (10.Wierzbicka-Patynowski I. Schwarzbauer J.E. J. Cell Sci. 2003; 116: 3269-3276Crossref PubMed Scopus (392) Google Scholar). Fibronectin matrices are essential for embryonic development, wound healing, and tumorigenesis (11.Wu C. Bauers R.L. Juliano R.L. McDonnell J.A. J. Biol. Chem. 1993; 268: 21883-21888Abstract Full Text PDF PubMed Google Scholar) and as such are both spatially and temporally regulated (11.Wu C. Bauers R.L. Juliano R.L. McDonnell J.A. J. Biol. Chem. 1993; 268: 21883-21888Abstract Full Text PDF PubMed Google Scholar). Thus, it is crucial to discern the mechanisms by which a fibronectin matrix is deposited and regulated. Fibronectin is bound to and regulated by cells at specific sites of cell-substratum adhesions that serve to link the ECM to the actin cytoskeleton via transmembrane integrin heterodimers (12.Geiger B. Bershadsky A. Curr. Opin. Cell Biol. 2001; 13: 584-592Crossref PubMed Scopus (485) Google Scholar). Forces due to integrin binding to ECM ligands cause the bundling of the actin cytoskeleton into tension-generating stress fibers. This tension also causes the clustering of integrins and associated cytoskeletal proteins, such as vinculin, paxillin, talin, c-Src, and focal adhesion kinase (FAK), into strong cell substratum adhesions termed focal contacts (or focal adhesions) (8.Geiger B. Bershadsky A. Pankov R. Yamada K.M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 793-805Crossref PubMed Scopus (1857) Google Scholar, 12.Geiger B. Bershadsky A. Curr. Opin. Cell Biol. 2001; 13: 584-592Crossref PubMed Scopus (485) Google Scholar, 13.Carragher N.O. Frame M.C. Trends Cell Biol. 2004; 14: 241-249Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 14.Frame M.C. Fincham V.J. Carragher N.O. Wyke J.A. Nat. Rev. Mol. Cell. Biol. 2002; 3: 233-245Crossref PubMed Scopus (277) Google Scholar). FAK plays a central role in cell-matrix adhesion and is also a substrate for Src (15.Cary L.A. Chang J.F. Guan J.L. J. Cell Sci. 1996; 109: 1787-1794Crossref PubMed Google Scholar). Ezzel et al. (16.Ezzell R.M. Goldmann W.H. Wang N. Parasharama N. Ingber D.E. Exp. Cell Res. 1997; 231: 14-26Crossref PubMed Scopus (229) Google Scholar) have shown that the ability of cells to spread out on a substratum correlates directly with the efficacy with which vinculin is coupled to focal contacts. Thus, calreticulin-overexpressing cells, which possess the greatest amount of vinculin, are well spread on the substratum, whereas the calreticulin underexpressers with the least amount of vinculin are poorly spread (4.Opas M. Szewczenko-Pawlikowski M. Jass G.K. Mesaeli N. Michalak M. J. Cell Biol. 1996; 135: 1913-1923Crossref PubMed Scopus (109) Google Scholar). Tyrosine phosphorylation has also been shown to regulate cell-substratum adhesion (17.Zhang Z. Izaguirre G. Lin S.Y. Lee H.Y. Schaefer E. Haimovich B. Mol. Biol. Cell. 2004; 15: 4234-4247Crossref PubMed Scopus (68) Google Scholar, 18.Ilic D. Kovacic B. Johkura K. Schlaepfer D.D. Tomasevic N. Han Q. Kim J.B. Howerton K. Baumbusch C. Ogiwara N. Streblow D.N. Nelson J.A. Dazin P. Shino Y. Sasaki K. Damsky C.H. J. Cell Sci. 2004; 117: 177-187Crossref PubMed Scopus (97) Google Scholar, 19.Katz B.Z. Romer L. Miyamoto S. Volberg T. Matsumoto K. Cukierman E. Geiger B. Yamada K.M. J. Biol. Chem. 2003; 278: 29115-29120Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 20.Ballestrem C. Erez N. Kirchner J. Kam Z. Bershadsky A. Geiger B. J. Cell Sci. 2006; 119: 866-875Crossref PubMed Scopus (84) Google Scholar); thus, the aim of this study was to investigate the role of protein tyrosine kinases, particularly c-Src, in the regulation of cell-substratum adhesion in cells differentially expressing calreticulin. c-Src is an important regulatory molecule of focal contacts and belongs to a family of nonreceptor protein-tyrosine kinases, including the ubiquitous kinases Yes and Fyn (21.Brickell P.M. Int. J. Exp. Pathol. 1991; 72: 97-108PubMed Google Scholar, 22.Parsons J.T. Parsons S.J. Curr. Opin. Cell Biol. 1997; 9: 187-192Crossref PubMed Scopus (355) Google Scholar). To determine how calreticulin, a Ca2+-binding ER-resident protein, influences cell spreading, fibronectin matrix deposition, and cell-substratum adhesion, we examined the activity of c-Src in cells differentially expressing calreticulin on both uncoated and fibronectin-coated surfaces. This study presents novel findings on the role of calreticulin in fibronectin matrix deposition and the regulation of c-Src activity via its influence on releasable Ca2+ from the ER. Cell Culture−Mouse L fibroblasts differing in calreticulin expression used for this study have been extensively characterized and described elsewhere (4.Opas M. Szewczenko-Pawlikowski M. Jass G.K. Mesaeli N. Michalak M. J. Cell Biol. 1996; 135: 1913-1923Crossref PubMed Scopus (109) Google Scholar, 6.Fadel M.P. Szewczenko-Pawlikowski M. Leclerc P. Dziak E. Symonds J.M. Blaschuk O. Michalak M. Opas M. J. Biol. Chem. 2001; 276: 27083-27089Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 23.Burns K. Duggan B. Atkinson E.A. Famulski K.S. Nemer M. Bleackley R.C. Michalak M. Nature. 1994; 367: 476-480Crossref PubMed Scopus (327) Google Scholar, 24.Mery L. Mesaeli N. Michalak M. Opas M. Lew D.P. Krause K.-H. J. Biol. Chem. 1996; 271: 9332-9339Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Sense and antisense cDNA were used to generate stable cell lines either over- or underexpressing calreticulin, respectively (23.Burns K. Duggan B. Atkinson E.A. Famulski K.S. Nemer M. Bleackley R.C. Michalak M. Nature. 1994; 367: 476-480Crossref PubMed Scopus (327) Google Scholar). Calreticulin-overexpressing cells displayed approximately twice as much calreticulin as control L fibroblasts, as determined by Western blot analysis. Calreticulin-underexpressing cells displayed approximately half as much calreticulin as control L fibroblasts. L fibroblasts were maintained in antibiotic free, high glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 100 μg/ml Geneticin. RNA Isolation and Northern Blotting−RNA extraction and Northern blotting were performed as described previously (23.Burns K. Duggan B. Atkinson E.A. Famulski K.S. Nemer M. Bleackley R.C. Michalak M. Nature. 1994; 367: 476-480Crossref PubMed Scopus (327) Google Scholar). The blots were normalized by probing with human glyceraldehde 3-phosphate dehydrogenase cDNA. Western Blotting−Protein samples from cellular lysates (40,000 cells/lane and 2 μg/lane for molecular weight markers) were subjected to SDS-PAGE and Western blotting as described previously (24.Mery L. Mesaeli N. Michalak M. Opas M. Lew D.P. Krause K.-H. J. Biol. Chem. 1996; 271: 9332-9339Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Primary antibodies were used at the following dilutions in TBST: anti-actin, 1:500 (Sigma); anti-calreticulin, 1:300 (25.Milner R.E. Baksh S. Shemanko C. Carpenter M.R. Smillie L. Vance J.E. Opas M. Michalak M. J. Biol. Chem. 1991; 266: 7155-7165Abstract Full Text PDF PubMed Google Scholar, 26.Fliegel L. Burns K. Opas M. Michalak M. Biochim. Biophys. Acta. 1989; 982: 1-8Crossref PubMed Scopus (58) Google Scholar, 27.Opas M. Dziak E. Fliegel L. Michalak M. J. Cell. Physiol. 1991; 149: 160-171Crossref PubMed Scopus (130) Google Scholar); anti-FAK (Tyr(P)397), 1:1000 (BioSource International); anti-fibronectin, 1:1000 (Sigma); anti-α5 and -β1 integrins, 1:500 (gift from Dr. B. Chan); anti-Src, 1:1000 (Upstate Cell Signaling); anti-Src (Tyr(P)418), 1:1000 (BioSource International); anti-active Src, 1:1000 (BioSource International); and anti-vinculin, 1:500 (Sigma). All horseradish peroxidase-conjugated secondary antibodies were used at a dilution of 1:10,000 (Jackson ImmunoResearch Laboratories). Isolation of the ECM fractions and identification of fibronectin in the ECM was carried out as previously described (28.Kornblihtt A.R. Pesce C.G. Alonso C.R. Cramer P. Srebrow A. Werbajh S. Muro A.F. FASEB J. 1996; 10: 248-257Crossref PubMed Scopus (172) Google Scholar). Briefly, the ECM was isolated by sequential washes with 1) PBS; 2) 3% Triton X-100 in PBS; 3) 50 mm Tris, pH 7.4, 10 mm MnCl2, 1 m NaCl; 4) 2% deoxycholate in 50 mm Tris, pH 8.8, 10 mm EDTA. All washes were carried out at room temperature for 3 min in the presence of 1 mm phenylmethylsulfonyl fluoride. The material remaining after the washes was considered to be ECM and was scraped in 1% SDS and boiled for 5 min prior to loading. Sample Preparation for Matrix-assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry (MALDI-TOF MS)−Aliquots of conditioned media were separated by SDS-PAGE. Proteins were visualized using Bio-Rad silver stain. For protein identification by MALDI-TOF MS, the band of interest was excised from the gel and placed in 1% acetic acid. Gel fragments were then washed with water, incubated in acetonitrile for 15 min at room temperature, and reduced in 100 mm ammonium bicarbonate containing 10 mm dithiothreitol for 30 min at 50 °C. After a second acetonitrile wash, the gel fragments were alkylated with 100 mm ammonium bicarbonate containing 55 mm iodoacetamide for 20 min at room temperature in the dark and washed in 100 mm ammonium bicarbonate. After a final acetonitrile wash, the gel fragments were dried by SpeedVac for 1 min. For trypsin digestion, the gel fragments were rehydrated in digestion buffer (50 mm ammonium bicarbonate, 5 mm CaCl2, and 12.5 ng/μl sequencing grade trypsin) (Promega) for 45 min on ice, and then incubated overnight at 37 °C. To extract tryptic fragments, after brief centrifugation, the liquid fraction was transferred to a fresh tube containing 100 mm ammonium bicarbonate for 30 min at 37 °C. After a second extraction, the liquids were pooled and purified by repeated passage through a ZipTipC18 (Millipore). The sample was eluted with 65% acetonitrile, 1% acetic acid and analyzed by MALDI-TOF MS by the Mass Spectrometry Laboratory, University of Toronto. Protein identification was performed using the ProFound data mining software (available on the World Wide Web at prowl.rockefeller.edu). Fluorescence-activated Cell Sorting−Cells were trypsinized, rinsed with PBS, and fixed with 3.7% formaldehyde in PBS for 10 min. After rinsing, the cells in suspension were incubated with anti-α5 or -β1 integrin primary antibodies for 30 min at room temperature under gentle agitation. The cells were then rinsed with PBS and incubated for 30 min at room temperature with secondary antibodies. The cells were rinsed with PBS, resuspended at equal densities, and analyzed using an EPICS Elite Cell Sorter (Beckman-Coulter). Fibronectin Patterned Substrata−Coverslips were coated with 10 μg/ml fibronectin from Sigma in PBS for 90 min at 37 °C and then rinsed in PBS. The coated surface was then scratched with a 1-ml pipette tip to remove fibronectin, creating areas of fibronectin carpet next to areas without. Immunolabeling and Microscopy−For immunolocalization, cells on coverslips were fixed in 3.7% formaldehyde in PBS for 10 min. After washing (three times for 5 min) in PBS, the cells were permeabilized for 2 min with 0.1% Triton X-100 in buffer containing 100 mm PIPES, 1 mm EGTA, and 4% (w/v) polyethylene glycol 8000 (pH 6.9) and then washed three times for 5 min in PBS and incubated with primary antibodies for 30 min at room temperature at the following dilutions in PBS: anti-fibronectin, 1:50, anti-α5 integrins, 1:50, anti-vinculin, 1:20; Texas Red phalloidin, 1:10. After washing in PBS, the cells were stained with appropriate fluorescent secondary antibodies (1:50 in PBS) for 30 min at room temperature. After the final wash, slides were mounted in Vinol 205S, which contained 0.25% 1,4-diazabicyclo-(2,2,2)-octane and 0.002% p-phenylenediamine or in Immuno Fluore (ICN Biochemicals). A Bio-Rad MRC-600 confocal fluorescence microscope equipped with a krypton/argon laser was used for fluorescence and phase-contrast microscopy. Tyrphostins, Radicicol, and Calcium Treatments−Before any treatments, equal numbers of cells were plated and grown overnight (12 h). Treatment times listed are those that follow this overnight attachment and growth. Cells were treated with 100 μm tyrphostin A1, A23, A25, or A63 for 24 h. The cells were then fixed and stained for immunofluorescence imaging as described above. For c-Src inhibition experiments, cells were treated with 5 μm radicicol (Sigma) overnight (12 h). The next day, cells were washed three times with PBS and replaced with fresh growth medium for 5 h. The cells were then collected either for Western blotting or fixed and stained for immunofluorescence imaging. Thapsigargin was used to reduce the concentration of Ca2+ in the ER. Cells were treated with 1 μm thapsigargin for 30 min at 37 °C and immediately collected for Western blotting of Src Tyr(P)418. For Western blotting of fibronectin and immunofluorescence of fibronectin deposition, cells were treated with 1 μm thapsigargin or 1 μm ionomycin for 2 h, followed by overnight growth in regular growth medium. Calcium Measurements−For measurements of intracellular Ca2+, cultured L cells, either in suspension or adherent to tissue culture dishes, were loaded with fura-2-AM (Molecular Probes) at a concentration of 3 μm, for 30 min at 37 °C. A Nikon inverted fluorescence microscope (Diaphot) optically interfaced to a dual wavelength excitation ratio fluorimeter (Photon Technology International) was used to estimate changes in intracellular [Ca2+], based on changes in fluorescence intensity at alternating 346/381 nm excitations. For bradykinin stimulation experiments, bradykinin (Sigma) was reconstituted in dimethyl sulfoxide and added to fura-2-loaded cells at a concentration of 2 μm to stimulate calcium release from intracellular stores. For the experiments using L cells plated on fibronectin, fura-2-loaded cells were trypsinized and suspended in a buffer solution containing HEPES, NaCl, KCl, glucose, MgSO4, Na2HPO4, and CaCl2. Cells were then seeded onto 32-mm diameter coverslips that were precoated with 10 μg/ml fibronectin (Sigma). Intracellular [Ca2+] was measured at 5, 15, and 30 min postplating. As a control, the experiment was performed with fura-2-loaded cells seeded onto untreated glass coverslips. L Fibroblasts Differentially Expressing Calreticulin Also Differ in Their Ability to Synthesize Fibronectin−Three stably transfected cell lines of mouse L fibroblasts were previously generated and extensively characterized (4.Opas M. Szewczenko-Pawlikowski M. Jass G.K. Mesaeli N. Michalak M. J. Cell Biol. 1996; 135: 1913-1923Crossref PubMed Scopus (109) Google Scholar, 6.Fadel M.P. Szewczenko-Pawlikowski M. Leclerc P. Dziak E. Symonds J.M. Blaschuk O. Michalak M. Opas M. J. Biol. Chem. 2001; 276: 27083-27089Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 23.Burns K. Duggan B. Atkinson E.A. Famulski K.S. Nemer M. Bleackley R.C. Michalak M. Nature. 1994; 367: 476-480Crossref PubMed Scopus (327) Google Scholar, 24.Mery L. Mesaeli N. Michalak M. Opas M. Lew D.P. Krause K.-H. J. Biol. Chem. 1996; 271: 9332-9339Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Cells with increased calreticulin expression level (overexpressers) or reduced calreticulin level (underexpressers) or mock-transfected cells with unchanged calreticulin level (control), as determined by Western blot analyses (23.Burns K. Duggan B. Atkinson E.A. Famulski K.S. Nemer M. Bleackley R.C. Michalak M. Nature. 1994; 367: 476-480Crossref PubMed Scopus (327) Google Scholar), were used in this study. Our previous studies showed that calreticulin influences the expression of vinculin, an intracellular component of focal contacts; thus, we examined here whether the extracellular face of focal contacts may also be affected by calreticulin. Our particular focus was on fibronectin, a major component of integrin-based cell substratum adhesions. Interestingly, we found that cells differentially expressing calreticulin differed in their ability to synthesize fibronectin. Using Northern blotting of total cellular lysates of L fibroblasts grown for 24 h, we found that calreticulin overexpressers had greater levels of fibronectin mRNA in comparison with either calreticulin underexpressers or control cells (Fig. 1A). Furthermore, calreticulin overexpressers contained the most intracellular fibronectin protein, whereas the underexpressers had the least amount and control cells were intermediate (Fig. 1B). The increased fibronectin protein in calreticulin overexpressing L fibroblasts was accompanied by an increase in the deposition of extracellular fibronectin into the matrix (Fig. 1B′). Next, we determined whether calreticulin’s effects were specific for fibronectin or if the secretion of other proteins was also affected. Conditioned media from the L fibroblast cell lines were collected at various time points and subjected to SDS-PAGE. The proteins were revealed by silver staining. Overall, there were no changes in the bulk outflow of proteins from cells differentially expressing calreticulin (Fig. 1, C and C′). However, there were differences in the secretion of a high molecular weight protein that migrated at the same mobility as fibronectin-positive controls, such that secretion was increased in calreticulin overexpressers and decreased in calreticulin underexpressers. Control cells secreted an intermediate amount of this protein (Fig. 1, C and C′). To identify this unknown protein, the indicated band (Fig. 1C, arrowhead) was excised from the gel and analyzed by MALDI-TOF MS as outlined under “Experimental Procedures.” The tryptic fingerprint was compared against a data base using the ProFound software. The first ranked retrieved protein from the list was fibronectin, with a molecular mass of 276.15 kDa. Of the 26 peptides measured, eight were found in the data base with sequence coverage of 5% (supplemental Table 1). Proteins from SDS-PAGE of conditioned media were also transferred to nitrocellulose before silver staining. Western blotting confirmed the identity of the secreted high molecular weight protein to be fibronectin (Fig. 1D). Taken together, these results show that calreticulin-overexpressing L cells have increased fibronectin mRNA and protein and secrete more fibronectin, which leads to a greater accumulation of it in the matrix. Importantly, this trend is also seen in HEK293 cells containing the calreticulin gene driven by a doxycycline-sensitive promoter following calreticulin induction, as well as in mouse embryonic fibroblast cell lines with normal calreticulin levels compared with calreticulin-null cells. 4S. Papp, M. P. Fadel, and M. Opas, unpublished data. In all cases, there is greater fibronectin expression in cells with greater calreticulin abundance. L Fibroblasts Overexpressing Calreticulin Assemble an Elaborate Fibronectin Matrix, whereas Calreticulin Underexpressers Exhibit Poor Fibronectin Deposition−We next examined if the relationship between the level of calreticulin expression and deposition of fibronectin into the ECM led to differential fibronectin matrix assembly. Using indirect immunofluorescence with anti-fibronectin antibodies on nonpermeabilized cells, we showed that crowded calreticulin overexpressers assembled an extensive, weblike fibronectin matrix after overnight growth in culture (Fig. 2). High magnification reveals extensive, thick fibronectin fibrils. Crowded L fibroblasts underexpressing calreticulin assembled a poor fibronectin matrix, which consisted of a loose arrangement of thin fibrils and patches. The fibronectin matrix assembled by control cells was intermediate between that of overexpressers and underexpressers (Fig. 2). Focal Contact Formation and Differential Localization of the Fibronectin Receptor in L Cells−Since the deposition and organization of a fibronectin matrix is cell-dependent and, more specifically, is remodeled by focal contacts, we next analyzed the adhesive architecture of cells. L fibroblasts double-labeled with fluorescently tagged phalloidin to reveal the F-actin cytoskeleton and an antibody to vinculin to show focal contacts were examined. Increased focal contact formation and a more robust stress fiber-containing actin cytoskeleton are evident in calreticulin-overexpressing cells (Fig. 3A). The calreticulin underexpressers are poorly spread and have very few focal contacts and no stress fibers. Quantification of the number of vinculin-containing focal contacts per cell reveals a significantly greater number of focal contacts in calreticulin overexpressers compared with control cells (Fig. 3B). Calreticulin underexpressers contain only a slight number of focal contacts, which are significantly lower than those in control cells (Fig. 3B). The fibronectin receptor in L fibroblasts is the α5β1 integrin heterodimer, and the extracellular domain of the α5 subunit is responsible for ligand binding (11.Wu C. Bauers R.L. Juliano R.L. McDonnell J.A. J. Biol. Chem. 1993; 268: 21883-21888Abstract Full Text PDF PubMed Google Scholar). We next wanted to determine if the difference in fibronectin matrix assembly in L cells was due to differential fibronectin receptor expression at the cell surface, since changes in calreticulin expression do not affect the total protein levels of α5β1 integrins in these cells (4.Opas M. Szewczenko-Pawlikowski M. Jass G.K. Mesaeli N. Michalak M. J. Cell Biol. 1996; 135: 1913-1923Crossref PubMed Scopus (109) Google Scholar, 6.Fadel M.P. Szewczenko-Pawlikowski M. Leclerc P. Dziak E. Symonds J.M. Blaschuk O. Michalak M. Opas M. J. Biol. Chem. 2001; 276: 27083-27089Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Fluorescence-activated cell sorting analysis revealed no differences in the surface expression of α5 and β1 integrins (Fig. 4A). However, the observed difference in fibronectin matrix deposition in L fibroblasts could result from differential receptor localization. Thus, immunolocalization of the α5 integrin subunit was performed, and distinct differences were detected in its localization to the cell surface in each L fibroblast cell line. Crowded calreticulin-overexpressing cells formed abundant α5 integrin-rich patches, which had the appearance of focal contacts (Fig. 4B). In calreticulin underexpressers, α5 integrin-positive patches were very scarce, and there was little focal contact formation (Fig. 4B). Control cells had an intermediate morphology with respect to α5 integrin and vinculin-containing focal contacts (Figs. 3A and 4B). Preformed Fibronectin Induces Cell Spreading and Increased Focal Contact Formation, with the Greatest Changes Occurring in Calreticulin Underexpressers−Since the calreticulin-underexpressing cells were unable to assemble a robust fibronectin matrix and showed poor focal contact formation, we asked if an exogenous preadsorbed fibronectin matrix would rescue the poor adhesive phenotype of these cells. Cells were grown overnight on precoated fibronectin substrata, and were imaged by confocal microscopy following double label immunolocalization for vinculin and fibronectin. This time of growth in vitro allowed for imaging of both preformed fibronectin matrix remodeling and initial stages of fibronectin matrix deposition on glass. To visualize changes in cellular morphology and adhesiveness due to fibronectin, we imaged the cells at the interface between the fibronectin carpet and fibronectin-free bare glass, positioned so as to fit into a single microscopic field of view. On glass, calreticulin-overexpressing cells were well spread, made many vinculin-positive focal contacts, and deposited fibronectin, whereas the calreticulin underexpressers were poorly spread with few, if any, focal contacts (Figs. 3A and 5A). In stark contrast, calreticulin underexpressers on fibronectin carpets spread extensively, and vinculin staining revealed a dramatic increase in the number of vinculin-containing focal contacts in calreticulin underexpressers" @default.
• W2000788954 created "2016-06-24" @default.
• W2000788954 creator A5022877139 @default.
• W2000788954 creator A5051002562 @default.
• W2000788954 creator A5054290782 @default.
• W2000788954 creator A5071277216 @default.
• W2000788954 creator A5076461409 @default.
• W2000788954 date "2007-06-01" @default.
• W2000788954 modified "2023-09-29" @default.
• W2000788954 title "Calreticulin Affects Fibronectin-based Cell-Substratum Adhesion via the Regulation of c-Src Activity" @default.
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